Deoxycytidine Kinase Gene Research Articles

Synergistic inhibition of pancreatic adenocarcinoma cell growth by trichostatin A and gemcitabine

We investigated the ability of the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) to interact with gemcitabine (GEM) in inducing pancreatic cancer cell death. The combined treatment with TSA and GEM synergistically inhibited growth of four pancreatic adenocarcinoma cell lines and induced apoptosis. This effect was associated with the induction of reactive oxygen species (ROS) by GEM, increased expression of the pro-apoptotic BIM gene by both TSA and GEM and downregulation of the 5′-nucleotidase UMPH type II gene by TSA. The expression of other genes critical for GEM resistance (nucleoside transporters, deoxycytidine kinase, cytidine deaminase, and ribonucleotide reductase genes) was not affected by TSA. The functional role of ROS in cell growth inhibition by GEM was supported by (i) a significantly reduced GEM-associated growth inhibition by the free radical scavenger N-acetyl- l-cysteine, and (ii) a positive correlation between the basal level of ROS and sensitivity to GEM in 10 pancreatic cancer cell lines. The functional role of both Bim and 5′-nucleotidase UMPH type II in cell growth inhibition by TSA and GEM was assessed by RNA interference assays. In vivo studies on xenografts of pancreatic adenocarcinoma cells in nude mice showed that the association of TSA and GEM reduced to 50% the tumour mass and did not cause any apparent form of toxicity, while treatments with TSA or GEM alone were ineffective. In conclusion, the present study demonstrates a potent anti-tumour activity of TSA/GEM combination against pancreatic cancer cells in vitro and in vivo, strongly supporting the use of GEM in combination with an HDAC inhibitor for pancreatic cancer therapy.

Prediction of the Chemotherapeutic Efficacy for Advanced Pancreatic Cancer By Focused DNA Array Analysis Using Endoscopic Ultrasound-Guided Fine-Needle Aspiration

Background: Patients with unresectable pancreatic cancers have extremely poor prognosis. The ineffective anti-cancer drugs, which cause adverse effect alone, should not be given to the patient with this dismal disease. It is an urgent investigational subject to predict the effectiveness of anti-cancer agent against pancreatic cancer prior to treatment. We examined the usefulness of Focused DNA Array (FDA) analysis using the pancreatic cancer tissue obtained by endoscopic ultrasound-guided fine-needle aspiration biopsy (EUS-FNA) for the prediction of chemotherapeutic effect. Methods: Tissues from 21 unresectable pancreatic cancers were obtained by EUS-FNA using the 19 or 22-gauge needle. Total mRNA was harvested from each sample, and cDNA was composed by reverse transcription polymerase chain reaction (RT-PCR). Expression of RNA was evaluated using FDA which was restricted to 133 genes including drug-resistance related genes. The amount of RNA was expressed as the relative value to house keeping gene. Result: Gemcitabine (GEM) was given to 17 patients. One course of GEM regimen composed 1000 mg/m2 weekly administration for three weeks following one week rest. Eleven patients completed 3 courses or more of this regimen were divided into the following two groups. Three patients with PR and three patients with tumor marker reduction by 50% or more were classified as responders. The other five patients were classified as non-responders. By the FDA, we measured the mRNA expression of dCMP deaminase (DCD) and RRM1 genes, which may act as GEM-resistant factors, and deoxycytidine kinase (DCK) gene, which may act as GEM-sensitive factor. Our gene scoring was defined as following. When the mRNA amount was half or less in GEM-resistant factors or twice or more in GEM-sensitive factor, compared to the mean amount of corresponding mRNA in all 21 tissues, one point was added to the gene score. When the mRNA amount was twice or more in GEM-resistant factors or half or less in GEM-sensitive factor, one point was subtracted from the gene score. In six responders, four tumors showed +1 (gene score) and two did +2. In five non-responders, one tumor indicated −1, three did zero point, and one did +1. When the gene score of +1 or more point was defined as a predictive indicator for GEM-efficacy, the sensitivity, specificity and accuracy were 100%, 80% and 91%, respectively. Conclusion: The FDA chip is loaded with many other genes related to 5-fluorouracil, cisplatin, molecular targeting agents, and so on, besides GEM. The gene analysis using a sample taken by EUS-FNA might be useful to predict the chemothrapeutic efficacy for patients with unresectable pancreatic cancer.

Multicapillary Electrophoresis Analysis of Single-Nucleotide Sequence Variations in the Deoxycytidine Kinase Gene

Investigation of the genetic background of complex traits is the focus of recent interest, as several common diseases or the individual response to treatments of various illnesses have not yet been explored. These studies require the development and implementation of reliable and large-scale genotyping methods. In this report, we introduce an efficient technique based on PCR-restriction fragment length sequence variation technique for the analysis of the -360CG and -201CT single-nucleotide sequence variations in the deoxycytidine kinase gene. A multicapillary gel electrophoresis instrument was used for the size determination of the generated DNA fragments. A healthy Hungarian population of 100 individuals was investigated to determine allele and genotype frequencies for the 2 sequence variations of interest. We found that the occurrence of the minor allele is rather low, i.e., the frequency of both the -360G and -201T variants is 1%. Our technique can readily facilitate the analysis of these important sequence variations in other ethnic groups to clarify the role of these sequence variations in conjunction with arabinosylcytosine treatment in acute myeloid leukemia.

Novel deoxycytidine kinase gene polymorphisms: a population screening study in Caucasian healthy volunteers

Deoxycytidine kinase (DCK) is the rate-limiting enzyme of the intracellular phosphorylation of nucleoside anticancer drugs, including gemcitabine and beta-arabinofuranosylcytosine, to their active triphosphates. This study was performed to assess the occurrence and frequency of DCK polymorphisms in a predominantly Caucasian population and to choose candidate polymorphisms for subsequent functionality studies. All seven DCK exons and the promoter region were sequenced from 100 healthy volunteers (79 females and 21 males). With respect to ethnicity, the study cohort comprised 93 Caucasian, one Asian, one African, and five mixed-race individuals. Six novel single nucleotide polymorphisms (SNPs) were found (-243G>T, -135G>C, 261G>A, 364C>T, 727A>C, IVS6+41T>A). Two SNPs are nonsynonymous and lead to changes in the amino acid sequence [C364T in exon 3 (P121S) and A727C in exon 6 (K242Q)]. The presence of the linked promoter polymorphism -360C>G/-201C>T was confirmed in Caucasians, but was less frequent than what has been reported from Asians (allele frequencies 2 versus 15.6%). The most prevalent haplotype was the wild-type plus IVS6+41TT (85.8%). This study found novel DCK polymorphisms, including nonsynonymous SNPs, in exons 3 and 6. A comparison of the data obtained in this study with those reported in a previous study on Asians [Shi et al. (2004) Pharmacogenetics 14:759-768] illustrates marked inter-ethnic differences in the occurrence and frequency of DCK polymorphisms. The higher allelic frequency of the promoter polymorphism -C360G/-C201T in Asians than in Caucasians might predispose Asians to nucleoside drug-associated toxicity. These data will be used to assess the effect of DCK candidate SNPs (promoter, exons 3 and 6) in patients receiving gemcitabine anticancer treatment.

Haplotyping of the deoxycytidine kinase gene by multicapillary electrophoresis

Haplotyping of the deoxycytidine kinase gene by multicapillary electrophoresis

5′-Nucleosidase and Deoxycytidine Kinase Gene Expressions in High-Risk Myelodysplastic Syndrome (MDS).

Various chemotherapeutic regimens including cytarabine (ara-C) for MDS patients have been less effective than those for AML patients. Althogh alterations in the expression of genes involved in ara-C metabolism have been reported to be related to the clinical outcome in AML patients, there is no report about MDS. Deoxycytidine kinase (dCK) is the rate-limiting enzyme in this activation process being the first phosphorylation of ara-C. Cytoplasmic 5′-nucleotidase (5′-NT) dephosphorylates ara-CMP. The concentration of the intracellular ara-CTP, active form of ara-C, depends on the activities of two enzymes. To determine whether the level of expression of 5′-NT and dCK is implicated in clinical outcome in patients with high-risk MDS (RAEB/RAEB-t), we analyzed the mRNA expression of these at diagnosis in bone marrow (BM) cells of patients with RAEB/RAEB-t using real-time PCR (rt-PCR).Materials and Methods: BM cells obtained from 22 patients (male: female=16:6) with untreated high-risk MDS. The mean age of all patients was 66.4±7.9 y. 7 patients (31.8%) had RAEB-t subtype and 15 (68.2%) patients RAEB subtype of the FAB classification. Quantitative rt-PCR of 5′-NT or dCK mRNA of BM cells in patients and 12 healthy volunteers was performed by LightCycler using β-actin as housekeeping control. For each sample, a ratio of 5′-NT or dCK value/β-actin value was calculated and considered as the level of 5′-NT or dCK mRNA expression.Results: 15 of patients received ara-C containing regimen (7 patients had supported care only). Complete remission was obtained in 7 patients. Other 8 patients were chemoresistant. The median overall survival (OS) of all patients was 22.9 months (range 2.0–91.0), and that of patients with chemotherapy was 20.0 (7–91). The median post-chemotherapy survival (PCS) was 15.0 months (5.0–43.0). At diagnosis, the mean and median expression level of 5′-NT mRNA in all patients was 1.56 (SD 1.61) and 1.36 (range 0.77–6.77), respectively (control: mean 0.23, SD 0.06 and median 0.67, range: 0.17–0.34, p<0.01). The expression of dCK mRNA did not show any significant difference between patients and control. Patients with chemotherapy whose BM cells were higher level of 5′-NT expression (greater than mean value) had shorter OS (14.0 months vs. 26.0 months, p<0.01) and shorter PCS (10.0 months vs. 17.0 months, p=0.011). [Display omitted] Conclusion: These data suggest that the expression level of 5′-NT mRNA might be a candidate for prognostic factor in RAEB/RAEB-t. Patients with an increase of 5′-NT mRNA at diagnosis show shorter overall survival and chemoresistance.

Genetic factors influencing Pyrimidine-antagonist chemotherapy

Pyrimidine antagonists, for example, 5-fluorouracil (5-FU), cytarabine (ara-C) and gemcitabine (dFdC), are widely used in chemotherapy regimes for colorectal, breast, head and neck, non-small-cell lung cancer, pancreatic cancer and leukaemias. Extensive metabolism is a prerequisite for conversion of these pyrimidine prodrugs into active compounds. Interindividual variation in the activity of metabolising enzymes can affect the extent of prodrug activation and, as a result, act on the efficacy of chemotherapy treatment. Genetic factors at least partly explain interindividual variation in antitumour efficacy and toxicity of pyrimidine antagonists. In this review, proteins relevant for the efficacy and toxicity of pyrimidine antagonists will be summarised. In addition, the role of germline polymorphisms, tumour-specific somatic mutations and protein expression levels in the metabolic pathways and clinical pharmacology of these drugs are described. Germline polymorphisms of uridine monophosphate kinase (UMPK), orotate phosphoribosyl transferase (OPRT), thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD) and methylene tetrahydrofolate reductase (MTHFR) and gene expression levels of OPRT, UMPK, TS, DPD, uridine phosphorylase, uridine kinase, thymidine phosphorylase, thymidine kinase, deoxyuridine triphosphate nucleotide hydrolase are discussed in relation to 5-FU efficacy. Cytidine deaminase (CDD) and 5'-nucleotidase (5NT) gene polymorphisms and CDD, 5NT, deoxycytidine kinase and MRP5 gene expression levels and their potential relation to dFdC and ara-C cytotoxicity are reviewed.

Role of USF1 in the differential expression of the human deoxycytidine kinase gene in acute myeloid leukemia

Role of USF1 in the differential expression of the human deoxycytidine kinase gene in acute myeloid leukemia

Determinants of sensitivity and resistance to gemcitabine: the roles of human equilibrative nucleoside transporter 1 and deoxycytidine kinase in non-small cell lung cancer.

Gemcitabine is one of the most commonly used agents for lung cancer chemotherapy, but the determinants of sensitivity and/or resistance to this agent are not yet fully understood. In this study we used quantitative RT-PCR to examine the expression levels of human equilibrative nucleoside transporter 1 (hENT1) and deoxycytidine kinase (dCK) genes in non-small cell lung cancer (NSCLC) cell lines in relation to sensitivity and resistance to gemcitabine. The basal expression levels of hENT1 were significantly correlated with the IC50 values for gemcitabine (r =-0.6769, P = 0.0005), whereas dCK expression levels were not. In a highly gemcitabine-sensitive cell line, NCI-H23, the sensitivity to gemcitabine was inhibited by nitrobenzylmercaptopurine ribonucleoside (NBMPR), an inhibitor of hENT1, without significant modulation of hENT1 expression. These data suggest that hENT1 is associated with gemcitabine sensitivity in lung cancer. We also continuously exposed NCI-H23 cells to gemcitabine and subsequently established the drug-resistant clone H23/GEM-R, which showed a significant decrease of dCK expression; however, hENT1 expression was not altered in the continuously exposed sublines or in the resistant clone. We conclude that increased hENT1 expression is a determinant of gemcitabine sensitivity, while decreased dCK expression is associated with acquired resistance to gemcitabine in NSCLC cells. Thus, hENT1 and dCK might be useful as predictive markers for efficacy of gemcitabine therapy in NSCLC.

Cytotoxic activity of 2 ′,2 ′-difluorodeoxycytidine, 5-aza-2 ′-deoxycytidine and cytosine arabinoside in cells transduced with deoxycytidine kinase gene

Deoxycytidine nucleoside analogs must be first phosphorylated to become active anticancer drugs. The rate-limiting enzyme in this pathway is deoxycytidine kinase (dCK). Cells deficient in this enzyme are resistant to these analogs. To evaluate the potential of dCK to be used as suicide gene for deoxycytidine nucleoside analogs, we transduced both human A-549 lung carcinoma and murine NIH3T3 fibroblast cell lines with this gene. The dCK-transduced cells showed an increase in cytotoxicity to the analogs, cytosine arabinoside (ARA-C), and 5-aza-2 ′-deoxycytidine (5-AZA-CdR). Unexpectedly, the related analog, 2 ′,2 ′-difluorodeoxycytidine (dFdC), was less cytotoxic to the dCK-transduced cells than the wild-type cells. For the A-549-dCK cells, the phosphorylation of dFdC by dCK was much greater than control cells. In accord with the elevated enzyme activity, we observed a 6-fold increased dFdC incorporation into DNA and a more pronounced inhibition of DNA synthesis in the A-549-dCK cells. In an attempt to clarify the mechanism of dFdC, we investigated its action on A549 and 3T3 cells transduced with both cytidine deaminase (CD) and dCK. We reported previously that overexpression of CD confers drug resistance to deoxycytidine analogs. In this study, when the CD-transduced cells were also transduced with dCK they became relatively more sensitive to dFdC. In addition, we observed that dFdU, the deaminated form of dFdC, was cytotoxic to the A-549-dCK cells, but not the wild-type cells. Our working hypothesis to explain these results is that the mitochondrial thymidine kinase (TK2), an enzyme reported to phosphorylate dFdC, acts as an important modulator of dFdC-induced cell toxicity. These findings may further clarify the action of dFdC and the mechanism by which it induces cell death.

Molecular basis of 2',3'-dideoxycytidine-induced drug resistance in human cells.

Human monoblastoid cells (U937) grown in the presence of therapeutically relevant dideoxycytidine concentrations (0.1 microM) become resistant to the drug thanks to an altered deoxycytidine kinase. In this paper we show that deoxycytidine kinase mRNA is significantly reduced in drug-resistant U937 cells (U937-R) although the deoxycytidine kinase promoter is normal. Anumber of nucleotide deletions, insertions and substitutions was found in the coding region of deoxycytidine kinase gene. Several identified mutations result in truncated forms of the enzyme or in the introduction of stop codons: in one case a complete lack of exon 4 was found. Thus, the deoxycytidine kinase gene accumulates mutations at a very high rate, as already reported for other cytidine analogues (i.e. Ara C) suggesting that the design of new antiviral or anticancer drugs of the cytidine family should take into account the potential development of cell resistance as a critical factor in drug failure.

Conserved gene structure and transcription factor sites in the human and mouse deoxycytidine kinase genes

Deoxycytidine kinase (dCK) phosphorylates several anti-cancer and anti-viral nucleoside analogs. The enzyme is predominantly expressed in lymphoid tissues regulated by an unknown mechanism. We have cloned and sequenced the 20 kbp mouse dCK gene and ≈1.7 kbp of the 5′ flanking regions of both the human and mouse dCK genes. Five major inter-species conserved motifs were identified in the 5′ region including the transcription initiation region, an SP1 site and two closely located putative octamer transcription factor sites. Luciferase reporter experiments showed that the human dCK 5′ region efficiently initiated transcription but no tissue regulatory element could be identified.

Transduction of the human deoxycytidine kinase gene in rodent tumor cells induces in vivo growth retardation in syngeneic hosts

Deoxycytidine kinase (dCK) mediates the phosphorylation of nucleoside analogues that can be used as anti-cancer agents. We examined whether susceptibility of mouse colon carcinoma (Colon 26) and rat gliosarcoma (9L) cells to 1-β- d-arabiofuranosylcytonsine (AraC), a chemotherapeutic agent, can be increased after the tumor cells were transduced with the human dCK gene. Expression of the dCK gene in both cell lines conferred increased sensitivity in vitro to AraC. Although their proliferation rates in vitro remained the same as those of parental cells, tumor growth of the transduced cells in syngeneic host animals was unexpectedly retarded compared with that of respective parental cells. In contrast, the growth of the transduced cells was not different from that of parental cells, when they were inoculated in T cell-defective nude mice. A histological examination revealed infiltration of eosinophils into the dCK gene-transduced but not into parental Colon 26 tumor. These data suggest that a therapeutic gene, when expressed in xenogenic animals, can be a tumor antigen which is recognized by a host defense system.

In vitro leukemia cell models of Ara-C resistance

Cells of the human leukemia line K562 were continuously exposed to cytosine arabinoside (Ara-C) at increasing concentrations for 3 months. The resulting cell line, termed K562/AC, showed 48-fold resistance to Ara-C, compared with the parental K562 cells. The sensitivities of K562/AC to adriamycin (ADR), vincristine (VCR) and etoposide (VP16) were similar to those of parental K562. Gene analysis revealed that this cell line lacked expression of the deoxycytidine kinase (dCK) gene, which was evident in Ara-C-sensitive cells. As in K562 cells, multidrug resistance (MDR-1) and multidrug resistance protein (MRP) genes were not expressed in K562/AC. We also established an in vitro model of Ara-C resistance using phosphorothioate antisense oligonucleotides to dCK (dCK-AS). Treatment of K562 with dCK-AS caused decreased dCK expression and 6- to 10-fold increases in resistance to Ara-C, compared with that in cells treated with sense oligonucleotides to dCK (dCK-S) or in non-transfected cells. The cells described here may contribute to the study of a novel mechanism associated with Ara-C resistance, in which reduced dCK activity may play an important role.

Analysis of DNA methylation of the 5′ region of the deoxycytidine kinase gene in CCRF-CEM-sensitive and cladribine (CdA)- and 2-chloro-2′-arabino-fluoro-2′-deoxyadenosine (CAFdA)-resistant cells

DNA methylation of the CpG-rich 5′ region of the deoxycytidine kinase (dCK) gene is potentially involved in the suppression of the gene and the resistance of tumour cells to arabinosylcytosine (ara-C). 2-Chlorodeoxyadenosine (cladribine, CdA) and 2-chloro-2′-arabino-fluoro-2′-deoxyadenosine (CAFdA) are purine nucleoside analogues which are also phosphorylated by dCK. We observed a reduction in dCK activity in a number of CCRF-CEM-derived cell lines that are resistant to these drugs and hypothesized that this reduction is due to DNA methylation of the 5′ region of the dCK gene. The DNA methylation state was analyzed at the DNA sequence level after bisulfite modification of genomic DNA. The investigated region included 0.3 kb of DNA upstream to the start site of transcription, exon 1 and part of intron 1. Sensitive cells (CCRF-CEM/0) and three resistant cell lines (CCRF-CEM/CdA4000, CCRF-CEM/CAFdA100 and CCRF-CEM/CAFdA4000) were investigated. The region that was analyzed contained no methylated cytosine residues in the parental cell line CCRF-CEM/0 or in the resistant cell lines. Therefore, it is highly unlikely that DNA methylation plays a role in the suppression of dCK gene expression in these cell lines.

Expression of Deoxycytidine Kinase (dCK) Gene in Leukemic Cells in Childhood: Decreased Expression of dCK Gene in Relapsed Leukemia

Competitive RT-PCR was used to determine the quantitative variation in the expression of deoxycytidine kinase (dCK) gene in childhood leukemic cells. The degree of dCK gene expression varied over a 50-fold range. In two cases in which both primary and relapsed leukemic cells were analysed, decreased expression of dCK gene was found in relapsed leukemic cells. The sequence variation analysis using bisbenzimide/polyethylene glycol electrophoresis demonstrated no sequence alteration of dCK cDNA in all cases. These results indicate that the expression of dCK gene varies in patients and suggests decreased expression of the dCK gene as one of the mechanisms responsible for clinical resistance to ara-C.

Loss of Deoxycytidine Kinase Expression and Tetraploidization of HL60 Cells Following Long-Term Culture in 1,25-Dihydroxyvitamin D3

Development of drug resistance is a major problem in attempts to control the growth of neoplastic cell populations. The resistance can be either inherent or acquired by an exposure to a chemotherapeutic drug. The available models for study of these phenomena have not led to major improvements in the therapy for human cancers. Therefore, in order to develop a new model for such studies, we have exposed human myeloid leukemia cells HL60 to increasing concentrations of 1,25-dihydroxyvitamin D3(1,25D3) and characterized the emerging new phenotypes of these cells over a period of 4 years. During the stepwise development of resistance only cells which did not adhere to the flask walls were passaged. Beginning at 30 nM1,25D3the sublines became resistant to the differentiation-inducing and growth-retarding properties of 1,25D3even at 400 nM.Also, their growth rates in 1,25D3-free media increased. In addition, beginning at 40 nM1,25D3the sublines acquired resistance to 5-β-D-arabinocytosine (araC) due to the lack of expression of the deoxycytidine kinase gene. The araC-resistant sublines were also near-tetraploid, as judged by their DNA content. When grown in 1,25D3-free long-term culture the phenotype was essentially stable. The development of cross-resistance to araC during growth in the presence of an unrelated compound (i.e., 1,25D3) shows that in some instances an apparently inherent drug resistance may in fact be due to a metabolic defect resulting from an exposure to another agent.

In vitro-induced resistance to the deoxycytidine analogues cytarabine (AraC) and 5-aza-2'-deoxycytidine (DAC) in a rat model for acute myeloid leukemia is mediated by mutations in the deoxycytidine kinase (dck) gene.

The deoxycytidine kinase (dck) gene encodes the enzyme responsible for the metabolic activation of the antileukemic drugs cytosine arabinoside (AraC) and 5-aza-2'-deoxycytidine (decitabine, DAC). The dck locus was analyzed at the chromosomal and the molecular level in a model of rat leukemic cell lines, in which AraC and DAC resistance was induced, that was marked by dck deficiency. At the chromosomal level, karyotype analysis of metaphase spreads revealed the presence of an aberrant 2q + chromosome in the AraC-resistant cell line and a (Xq:11q) translocation in its subclone RA/7. The DAC-resistant lines were identical to the parental RCL/O. Fluorescence in situ hybridization on normal rat fibroblast metaphase spreads localized the rat dck gene to chromosome 14q21-q22, a region that was not involved in any of the observed karyotypic aberrations. Analysis at the molecular level revealed an identical rearrangement of the dck gene in the AraC-resistant cell line RCL/A and its subclone RA/7 that resulted in the absence of dck expression, as assessed by RT-PCR. No genomic rearrangements were observed in a DAC-resistant cell line RCL/D or in its subclone RD/1. However, detection of a single-stranded conformation polymorphism (SSCP) allowed the identification of a single C to G substitution (His to Gln) in the dck cDNA of the DAC-resistant RD/1 clone. The data demonstrate that exposure to AraC and DAC induces a resistant phenotype marked by functional dck deficiency that may be the consequence of mutations occurring in the dck gene.

In vitro-induced resistance to the deoxycytidine analogues cytarabine (AraC) and 5-aza-2?-deoxycytidine (DAC) in a rat model for acute myeloid leukemia is mediated by mutations in the deoxycytidine kinase (dck) gene

In vitro-induced resistance to the deoxycytidine analogues cytarabine (AraC) and 5-aza-2?-deoxycytidine (DAC) in a rat model for acute myeloid leukemia is mediated by mutations in the deoxycytidine kinase (dck) gene

A mapping study of 13 genes on human chromosome bands 4q11→q25

A chromosome band 4q21 gene (MLLT2, formerly called AF-4/FEL) involved in a reciprocal translocation with chromosome band 11q23 in t(4;11) acute leukemia has been cloned. To provide better definition of gene order and relationships in this region where MLLT2 resides, we used pulsed field gel electrophoresis (PFGE) to investigate 13 genes (including MLLT2) with physical locations in bands 4q11-->q25. Somatic cell hybrids derived from RS4;11, a leukemic cell line carrying the t(4;11)(q21;q23), were also used to localize genes in relation to MLLT2. Linkage of the interleukin 8 (IL8), albumin (ALB), and platelet factor 4 (PF4) genes was confirmed by NotI, SalI and SacII digests. The maximum distance between PF4 and ALB is 210 kb and between ALB and IL8 is 420 kb. The alcohol dehydrogenase, class I (ADH2, ADH3) gene cluster can be linked to the alcohol dehydrogenase, class III gene (ADH5) by SacII, NruI, and EagI digests. The maximum distance between them is 590 kb. Our study indicated that ALB, alpha-fetoprotein (AFP), PF4, beta-thromboglobulin (PPBP), GRO1 (encoding a cytokine also called melanoma growth-stimulatory activity), and IL8 genes can be physically linked. In this study the gamma-interferon induced protein 10 (INP10), bone morphogenetic protein 3 (BMP3), annexin III (ANX3), KIT, amphiregulin (AREG), immunoglobulin J polypeptide (IGJ), deoxycytidine kinase (DCK) and MLLT2 genes were not linked to one another or to the above two groups of genes. Our analysis using somatic cell hybrids combined with previous reports demonstrated that the ADH gene cluster is telomeric to MLLT2 and KIT, ALB, AFP, PF4, beta TG, GRO1, IL8, ANX3, AREG and DCK are centromeric to MLLT2.